Arrestins play an essential role in regulating signaling and trafficking of G protein-coupled receptors (GPCRs). Visual arrestins (arrestin-1 and 4) specifically interact with opsins while non-visual arrestins (arrestin-2 and 3) interact with multiple GPCRs and also bind phosphoinositides, components of the endocytic machinery (clathrin and beta2-adaptin), and various signaling molecules (Src family members and iMAP kinases). Crystallographic analysis reveals that arrestin-1 and 2 have a similar basal structure containing N-terminal and C-terminal domains joined by a polar core. Disruption of the polar core is thought to occur upon receptor binding and result in an "active" conformation that regulates arrestin interaction with various proteins. The structural basis for arrestin activation as well as for arrestin interaction with GPCRs, phosphoinositides, endocytic components, and signaling molecules remains largely unknown. In this application, we propose to use X-ray crystallography and various biochemical and cell biological strategies to analyze the structural and molecular basis for arrestin-2 activation and interaction with components of the endocytic machinery. The specific objectives are to: 1. Perform crystallographicanalysis of arrestin complexed with phosphoinositides, clathrin, and beta2-adaptin. Although structure/function analysis has provided important insight into the domains that mediate arrestin interaction with phosphoinositides and endocytic components, a detailed picture of the binding interfaces of these components is lacking. We propose to crystallize and solve structures of wild-type and activated arrestin-2 in complex with inositol hexakisphosphate, clathrin, and beta2-adaptin. High-resolution crystallographic analysis of these complexes should provide structural insight into: 1) the binding interfaces of arrestin-2 with phosphoinositides, clathrin, and beta2-adaptin; 2) the mechanism of arrestin-2 activation; and 3) the mechanism of assembly of a multi-protein endocytic complex. 2. Characterize the biology of arrestin interaction with phosphoinositides, clathrin, and beta2-adaptin. The dynamics of arrestin interaction with clathrin-coated pits is complex and involves multiple protein-protein and protein-lipid interactions. We will use insight from our structural analysis to further define the mechanistic basis for arrestin-mediated regulation of GPCR endocytosis. These studies will utilize various biochemical, molecular, and cell biological strategies to probe the interaction of arrestins with endocytic components and elucidate the function of such interactions in GPCR trafficking. Overall, these studies should provide structural insight into differences in the basal and activated forms of arrestin-2 and provide a mechanistic basis for how non-visual arrestins function as GPCR-regulated scaffolding proteins.